Analysis of single nucleotide polymorphisms using a nicking endonuclease

Abstract

A method of genome analysis is provided. In certain embodiments, the method comprises: a) contacting a double-stranded genomic DNA with a site-specific nicking endonuclease that recognizes a sequence comprising a single nucleotide polymorphism (SNP), in which the endonuclease nicks the genomic DNA at a nick site only if a first allele of the SNP is present; b) denaturing the genomic sample; c) contacting the denatured genomic sample with an array comprising a first probe and a second probe, in which nicking results in less binding of the denatured sample to the first probe relative to a sample that is not nicked; and d) comparing the amount of hybridization to the first probe to the amount of hybridization to said second probe, in which decreased binding of the denatured genomic samples to the first probe relative to the second probe indicates that the first allele of the SNP is present.

1. A method comprising:
 a) contacting a sample comprising double-stranded genomic DNA with a site-specific nicking endonuclease that recognizes a nucleotide sequence that comprises a single nucleotide polymorphism (SNP) in said double stranded genomic DNA to provide a contacted genomic sample, wherein said endonuclease nicks said genomic DNA at a nick site that is proximal to said SNP only if a first allele of said SNP is present; 
 b) denaturing said contacted genomic sample to provide a denatured genomic sample; 
 c) contacting under hybridization conditions said denatured genomic sample with an array of probes comprising a first probe and a second probe, wherein:
 i) said first probe hybridizes to a nucleotide sequence in a first strand of said genomic DNA, wherein said nucleotide sequence comprises said SNP; and 
 ii) said second probe hybridizes to a nucleotide sequence in a second strand of said genomic DNA, wherein said nucleotide sequence comprises said SNP; and 
 iii) nicking at said nick site by said site-specific nicking endonuclease results in less binding of said denatured genomic sample to said first probe relative to a denatured genomic sample that is not nicked at said nick site; and 
 
 d) comparing the amount of hybridization to said first probe to the amount of hybridization to said second probe, wherein decreased binding of said denatured genomic samples to said first probe relative to said second probe indicates that said first allele of said SNP is present. 
 
     
     
       2. The method of  claim 1 , wherein said comparing step d) provides a ratio of the amount of hybridization to said first probe to the amount of hybridization to said second probe. 
     
     
       3. The method of  claim 2 , wherein said ratio of about 1:2 indicates a genotype that is heterozygous for said SNP. 
     
     
       4. The method of  claim 2 , wherein said ratio of about 1:1 or 0:1 indicates a genotype that is homozygous for said SNP. 
     
     
       5. The method of  claim 1 , further comprising measuring a copy number of a sequence comprising said SNP based on the amount of hybridization to said first probe or to said second probe. 
     
     
       6. The method of  claim 1 , wherein said first probe and said second probe have T m s that are within 10° C. of one another. 
     
     
       7. The method of  claim 1 , wherein said double-stranded genomic DNA is contacted with at least two site-specific nicking endonucleases, wherein said at least two site-specific nicking endonucleases recognize different nucleotide sequences comprising an SNP. 
     
     
       8. The method of  claim 1 , wherein said array comprises multiple probe sets, wherein each set of said multiple probe sets comprises a first probe and a second probe and is for detecting a different SNP. 
     
     
       9. The method of  claim 1 , further comprising contacting a reference sample to said array of probes, wherein said nucleotide sequence comprising said SNP in said reference sample is known. 
     
     
       10. The method of  claim 1 , wherein said contacting step a) further comprises contacting said double-stranded genomic DNA with a restriction endonuclease. 
     
     
       11. The method of  claim 1 , wherein said double-stranded genomic DNA is from a human. 
     
     
       12. The method of  claim 11 , wherein said first probe or said second probe is complementary to an SNP allele that is associated with a known disease or condition. 
     
     
       13. The method of  claim 1 , wherein said double-stranded genomic DNA is isolated from a genomic sample. 
     
     
       14. The method of  claim 1 , wherein said double-stranded genomic DNA is amplified from a genomic sample. 
     
     
       15. The method of  claim 1 , further comprising fragmenting said genomic DNA prior to contacting said sample with said site-specific nicking endonuclease. 
     
     
       16. The method of  claim 1 , wherein said first and second probes hybridize with a contiguous sequence of at least 20 nucleotides in length comprising said SNP and wherein nick site of said site-specific nicking endonuclease is located at or near the middle of said contiguous sequence. 
     
     
       17. An array comprising a first probe and a second probe, wherein
 a) said first probe is complementary to a nucleotide sequence in a first strand of a double-stranded genomic DNA, wherein said nucleotide sequence comprises a recognition site of a site-specific nicking endonuclease; 
 b) said second probe is complementary to a nucleotide sequence in a second strand of said double-stranded genomic DNA, wherein said second strand is complementary to said first strand; 
 c) said recognition site comprises an SNP; and 
 e) nicking of said first strand of said double-stranded genomic DNA by said site-specific nicking endonuclease results in less binding of said first strand to said first probe relative to an un-nicked strand. 
 
     
     
       18. The array of  claim 17 , wherein said first probe and said second probe have T m s that are within 10° C. of one another. 
     
     
       19. A kit for sample analysis comprising:
 a) a site-specific nicking endonuclease that recognizes a nucleotide sequence comprising a SNP; and 
 b) an array of  claim 17 . 
 
     
     
       20. The kit of  claim 19 , further comprising a reference genomic DNA, wherein said SNP in said reference genomic DNA is known.